Comparative genome analysis of colistin-resistant Escherichia coli harboring mcr isolated from rural community residents in Ecuador and Vietnam

The spread of colistin-resistant bacteria among rural community residents of low- and middle-income countries is a major threat to community health. Although the mechanism of the spread of colistin-resistant bacteria in communities is unknown, geographic and regional characteristics may influence it. To elucidate the spread mechanism of colistin-resistant bacteria, we analyzed the genomes of colistin-resistant Escherichia coli isolated from Vietnam and Ecuador residents, which are geographically and socially different. Stool specimens of 139 and 98 healthy residents from Ecuador and Vietnam rural communities, respectively, were analyzed for colistin-resistant E. coli with mcr. Its prevalence in the residents of all the communities assessed was high and approximately equal in both countries: 71.8% in Ecuador and 69.4% in Vietnam. A phylogenetic tree analysis revealed that the sequence type of colistin-resistant E. coli was diverse and the major sequence types were different between the two countries. The location of mcr in the isolates showed that the proportion of chromosomal mcr was 35.1% and 8.5% in the Vietnam and Ecuador isolates, respectively. Most of these chromosomal mcr genes (75%–76%) had an intact mcr-transposon Tn6330. Contrastingly, the replicon types of the mcr-carrying-plasmids were diverse in both countries, but almost all belonged to IncI2 in Ecuador and IncX1/X4 in Vietnam. Approximately 26%–45% of these mcr-plasmids had other resistance genes, which also varied between countries. These results suggest that although the overall profile of the colistin-resistant E. coli isolates is diverse in these countries, the phylogenesis of the isolates and mcr-carrying plasmids has regional characteristics. Although the contributing factors are not clear, it is obvious that the overall profile of colistin-resistant bacteria dissemination varies between countries. Such different epidemic patterns are important for establishing country-specific countermeasures against colistin-resistant bacteria.


Introduction
The recent emergence and spread of carbapenem-resistant Gram-negative bacteria poses a major threat to human health [1].Despite their toxicity, polymyxins are used as a valuable therapy for treating infections caused by these bacteria [2,3].Colistin (polymyxin E) belongs to the family polymyxin and has been available in clinical practice since the 1960s; so far, colistin is still the primary treatment for infections caused by carbapenem-resistant Gram-negative pathogens [4].Colistin resistance was known by chromosomal mutations relating to the structural alteration of lipid A in the lipopolysaccharide (LPS), a binding target of colistin [5,6].Common mutations that occurred with colistin resistance in Escherichia coli were reported in associated genes like pmr, pho, and mgrB.Recently, the mobile resistance genes known as mcr genes, which encode a phosphoethanolamine transferase, have been identified.This enzyme modifies the lipid A structure, subsequently reducing the binding affinity of colistin to LPS [6].It has been identified as a primary mechanism for colistin resistance due to its mobility.Thus far, at least ten mcr variants from mcr-1 to mcr-10 have been described globally [7,8].It is widely accepted that the major mechanism of the horizontal spread of colistin-resistant bacteria is the transfer of the mobile mcr genes among bacteria [9,10].
Colistin-resistant mcr-mediated E. coli is present globally, especially in Asia, Europe, and South America [11,12].In a previous study, we found that the prevalence of colistin-resistant E. coli carrying mcr-1 isolated from the residents of a community of Vietnam was 69.3% [13].However, knowledge regarding the spread of colistin-resistant bacteria with mcr in other communities/regions is limited; particularly, studies comparing the effects of geographical and cultural backgrounds of residents of different countries on the spread of these pathogens are lacking.
Colistin-resistant E. coli carrying mcr has also been found in high frequencies in domestic livestock in local communities of Vietnam, as reported in our previous studies [10,14].In another study, the prevalence of mcr-1 and mcr-3 detected in human and animal stools in a rural community of Vietnam were higher than that in previous reports [15].The same situation was observed in a rural small-scale farm in Ecuador, where mcr-mediated colistin-resistant E. coli was widespread [16].Due to the high prevalence of colistin-resistant E. coli carrying mcr in livestock as well as the possibility of horizontal transfer among various hosts, there is a high risk of the spread of resistant bacteria in the human community, given that animals are used for food production [17].Such a spread among communities and regions may be influenced by the people's lifestyles, which may also affect colistin-resistant bacteria epidemic patterns.Ecuador is located in Latin America with an upper middle-income level, whereas Vietnam is a lower middle-income country in Southeast Asia [18,19].Although both Ecuador and Vietnam are tropical countries with characteristics of high temperature and humidity, the average temperature in Vietnam was observed to be higher than in Ecuador.In addition, the primary agricultural practices of the two countries differ significantly.These two countries are also different regarding religions, while 85.6% of Ecuador residents belong to Christianity, 73.2% of Vietnam residents are nondenominational [20].Therefore, it is important to examine the dissemination of colistin-resistant bacteria in geographically and culturally different regions to elucidate the mechanism underlying the epidemic spread of colistin-resistant bacteria.Therefore, this study aimed to assess the spread of colistin-resistant bacteria in Vietnam and Ecuador using a comparative genome analysis approach.

Specimen collection
Stool samples provided by human residents of the communities were used as specimens in the study.Specimen collection was conducted in one community in Vietnam from November 1, 2017 to February 28, 2018 and in two communities in Ecuador from February 15 to March 8, 2019.These communities were relatively small rural villages.The major exclusion criterion for participants was antibiotic use in the last six months.A total of 237 healthy residents of Nguyen Xa in the Thai Binh province, Vietnam, Santo Domingo in the Pacific Coastal, and Puyo in the Amazon of Ecuador were enrolled.The participant characteristics are shown in Table 1.One stool specimen was obtained from each resident.Specimen collection and bacterial isolation in Vietnam have previously been reported [13]; the isolated bacteria were used for analysis in this study.

Isolation of colistin-resistant E. coli
The stool specimens were directly inoculated onto selective agar medium CHROMagar™ COL-APSE plates (CHROMagar, Paris, France) for isolation of the colistin-resistant Gramnegative bacteria.The resulting E. coli-like colonies, which showed pink to red color on the agar, were isolated.

Characterization of the isolates
The colistin-resistant E. coli isolates were identified via biochemical test using the analytical profile index (API) 20E system (bioMerieux, Marcy-l'E ´toile, France) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) (bioMe ´rieux Japan, Tokyo, Japan).

Multiplex PCR for colistin-resistant genes
Bacterial DNA was extracted by boiling the bacterial suspension for 10 min in tris (hydroxymethyl) aminoethane-ethylenediaminetetraacetic acid (EDTA) buffer.The PCR screening for mcr-1 to mcr-5 was performed using the QIAGEN Multiplex PCR Plus kit (Qiagen, Hilden, Germany).The primers, PCR conditions, and electrophoresis was done according to a method described previously [21].

Antimicrobial susceptibility testing
The colistin minimum inhibitory concentration (MIC) of colistin-resistant E. coli was measured using an agar dilution method as previously described [22].MICs �2 μg/mL were

Whole-genome DNA sequencing and analysis
DNA extraction.The genomic DNA of the isolates was extracted using NucleoBond HMW DNA (Macherey-Nagel, Du ¨ren, Germany) according to the manufacturer's protocol.The DNA was quantified using Qubit Double-Stranded DNA High-Sensitivity Assay Kits (Thermo Fisher Scientific, Waltham, MA, USA) and quality was assessed using a NanoDrop (Thermo Fisher Scientific).The A 260/280 and A 260/230 ratios of the DNA samples were within the range mentioned in the manufacturer's instructions for genome sequencing.
Library preparation and sequencing.Whole genome sequences were obtained using the DNBSEQ-G400FAST (MGI Tech, Shenzhen, China) and MinION Mk1C sequencer (Oxford Nanopore Technologies, London, UK).Short-read sequencing using the DNBSEQ-G400RS High-throughput Sequencing Set (MGI Tech) was performed by a commercial vendor (Genome-Lead Co., Kagawa, Japan).For the long-read sequencing, which was carried out using a MinION, the high molecular weight DNA of each isolate was barcoded using the Rapid Barcoding Kit (Oxford Nanopore Technologies) and was pooled into one PCR tube.To prepare the library, sequencing adapters were attached to the DNA sequences.The MinION flow cell (R9.4.1) was primed and loaded with the prepared library for a 24-h run on the Min-ION Mk1C.
De novo assembly.To obtain the complete genome of all tested isolates, a de novo hybrid assembly of both short-and long-reads was conducted using Unicycler 0.4.8, with default settings, on a supercomputer system and CLC Genomics Workbench 21.0.3.The short-read sequence was trimmed, and its quality was checked using fastp 0.20.1 and FastQC 0.11.9.The quality of long reads was verified using NanoPlot (http://nanoplot.bioinf.be/).In Unicycler, SPAdes v3.13.1 (with the maximum k-mer 127), Miniasm, Racon v.1.4.3, bowtie2 v.2.3.5, and SAMtools v.1.9were run for assembly, and the complete bacterial genome was polished using Pilon v.1.23.Using the CLC Genomics Workbench, de novo assembly of long reads was performed in the Long Read Support (beta) plugin, followed by polishing with short reads using the Polish with Reads (beta) tool.In this study, we obtained a total of 84 complete E. coli genomes (S1 and S2 Tables ).
Genome comparison.The similarity between a central reference sequence and others in this study was illustrated using BLAST Ring Image Generator (BRIG) 0.95 (http://brig.sourceforge.net).
Phylogenetic tree generation.The phylogenetic tree of the isolates was based on the single nucleotide polymorphism (SNPs) data generated using PARsnp version 1.7.4,run on multi-MUM as well as libMUSCLE aligner (https://github.com/marbl/parsnp),and constructed using iTOL (https://itol.embl.de).The initial tree was generated automatically (by default).

Statistics
The data of the number of residents carrying mcr-E.coli, the mcr-1 location, types of mcr-1transposon structures, dissemination of Inc-types of mcr-plasmids, and plasmids with antimicrobial-resistant (AMR) genes were compared among three groups of areas via the chi-square test and descriptive statistics using the statistical package for the social sciences (SPSS) version 20.0 software (IBM Corporation, Armonk, NY, USA).A p-value of <0.05 was considered statistically significant.

Prevalence of colistin-resistant E. coli carrying mcr in stool specimens of the residents
Results of the prevalence of mcr-positive colistin-resistant E. coli, which showed colistin MIC �4 μg/mL in healthy residents in Vietnam and Ecuador, are shown in Table 1.The prevalence of colistin-resistant E. coli with mcr-1 in the three communities of the two countries was similar, ranging from 69%-73%, with no significant difference.Notably, among selected isolates for whole genome sequencing, all 84 isolates carrying the mcr-1.1 gene, as shown in S1 and S2 Tables.

Phylogenetic analysis of colistin-resistant E. coli isolates among the communities
In this study, approximately half of the isolates from both countries, including 47 of 100 mcr-E.coli isolates in Ecuador and 37 of 68 mcr-E.coli isolates in Vietnam were randomly selected for sequencing.The complete genome was obtained for all 84 isolates that were sequenced (S1 and S2 Tables).The phylogenetic trees of colistin-resistant mcr-E.coli isolates from both countries were then generated using core-genome SNPs (Fig 1).
The isolates obtained from Ecuador residents were allocated to 30 sequence types (STtypes).Among them, two ST-types, ST48 and ST10, were dominant and three-ST206, ST2705, and ST6808-were more prevalent than the other ST-types, which appeared only once among the isolates.Based on the ST-types and the similarity among homologous sequences, the isolates were clustered into five major clades (Fig 1A).The first clade (clade 1) included four E. coli isolates; three of them were isolated in Puyo and one was from Santo Domingo with two ST6808 isolates.The second and third clades with three and four isolates mainly included ST2705 and ST206, respectively.The fourth and fifth clades represented two major branches among the Ecuador isolates, which comprised 12 and 13 strains with predominantly ST48 and ST10, respectively.As shown in Fig 1A, the colistin-resistant E. coli isolates from samples from both Santo Domingo and Puyo were diverse; however, 48.9% of them carried the IncI2 plasmid with mcr-1.
In Vietnam, 21 major ST-types, including ST165, ST206, ST48, and ST10, were detected among the 37 isolates, as in Ecuador.Five clades were clustered from these isolates.In contrast to Ecuador samples, ST165, ST206, and ST10 were the only STs in clades 1, 2, and 3, respectively.The fourth clade included three ST48 and the fifth clade included two ST101 isolates.Only three isolates (8.1%) carried the mcr-IncI2 plasmid among the Vietnam isolates (Fig 1B).
The phylogenetic trees of all the isolates obtained from Ecuador and Vietnam samples were analyzed together.As shown in S1 Fig, the colistin-resistant E. coli isolates from both countries were phylogenetically diverse and covered most branches.For instance, ST10, ST48, and ST206 were found in large numbers in both Ecuador and Vietnam samples.

mcr location
Since a high chromosomal mcr proportion of 35.1% was observed in Vietnam isolates [23], we used genome analysis to examine whether a similar chromosomal mcr prevalence also existed in Ecuador isolates.As shown in Table 2, only 14.8% of the Santo Domingo isolates carried mcr-1 on their chromosomes.All the Puyo isolates only harbored mcr-1 on the plasmid, and none of the isolates possessed chromosomal mcr.Thus, the chromosomal mcr proportion of the mcr-E.coli isolates in Ecuador was significantly lower than that in Vietnam (p = 0.006).However, a difference in chromosomal mcr ratios was not observed between communities within Ecuador (p = 0.204).
Contrastingly, no association was observed among the ST-types harboring chromosomal mcr in samples from both countries (Fig 1).

Transposon structure of mcr
Transposon structure is important for mcr transfer and stability.We compared the mcr transposon structures of all isolates.Results showed that the structure of the mcr-transposon was different between the chromosome and the plasmid of the isolates among all three communities.In the isolates from both Santo Domingo in Ecuador and Nguyen Xa in Vietnam, the intact mcr-transposon Tn6330 structure (type A), ISApl1-mcr1-PAP2-ISApl1, was dominantly located on the chromosome (Table 2).In contrast, the prevalence of the incomplete mcr-transposon structures, such as type B, ISApl1-mcr1-PAP2, and type C, mcr1-PAP2, was low on the chromosome.There was no isolate with type B in Santo Domingo samples and no isolate with type C in Nguyen Xa samples.
The mcr-transposon structure on the plasmids also differed between Ecuador and Vietnam isolates.As shown in Table 2, all the Vietnam isolates possessed plasmids carrying an incomplete mcr-transposon, such as type B or C.There was no plasmid with intact mcr-transposon type A. In contrast, many plasmids (39%-45%) in the isolates from both communities in Ecuador had an intact mcr-transposon type A. However, more than half of the mcr-transposons were type B, lacking a partial insertion sequence, ISApl1, or type C, which was completely lacking.
For further analysis of the mcr-transposon genetic characteristics, the genetic environment around the mcr-transposon on the chromosome was assessed.Of the 17 isolates with chromosomal mcr, 16 isolates carrying mcr-transposons with insertion sequence (IS), type A and B, were analyzed for their chromosomal insertion sites.The results showed that the insertion sites of the mcr-transposon on the chromosomes of all these isolates were surrounded by ATrich regions.A representative genetic environment map of the mcr-transposon on the 2018-11-1BCC chromosome of Vietnam is shown in S2 Fig.

mcr-plasmid incompatibility (Inc)-type
The Inc-type of the mcr-plasmid is important for plasmid transfer and spread.The regional Inc-type of mcr-carrying plasmids, which are the main cause of the spread of mcr-carrying colistin-resistant bacteria, was assessed.As shown in Table 3, among the three communities, the Inc-type of mcr-carrying plasmids was diverse and disseminated differently; moreover, the major mcr-carrying plasmid type differed between Ecuador and Vietnam isolates.I2-plasmids were significantly more frequent among the Ecuador isolates, at 50%-56%.In contrast, the P1 and X1/X4 plasmids were dominant in the Vietnam isolates, at 25%-29%.The hybrid plasmids carrying mcr were also dominant, ranging from 13%-35%, with no significant differences among the communities.Regarding the hybrid types, FIA/HIA/HIB was dominant in the Puyo isolates, while HI2/HI2A was dominant in the Nguyen Xa isolates (Table 4).The structures of I2-plasmids in both Ecuador and Vietnam isolates were compared; the results showed that the size and constituent genes were similar among these plasmids (Fig 2).Similarly, the sequences of other plasmid Inc-types, including X1, X4, and P1 mcr-plasmids, were compared.As shown in S3-S5 Figs, these plasmid Inc-types were also similar across communities and countries.

AMR genes on mcr-plasmids
Among the plasmids carrying mcr-1 from isolates, the presence of other AMR genes was assessed in Ecuador and Vietnam.As shown in Table 5 (refer to S1 and S2 Tables for details), the percentage of mcr-plasmids carrying no other AMR genes regardless of which community the isolate was from was higher than multi-AMR genes carrying plasmids, with 60%, 54.2%, and 73.9% in Puyo, Nguyen Xa, and Santo Domingo, respectively.However, mcr-carrying hybrid plasmids, which amalgamate two or more distinct Inc-type plasmid, in the isolates from both countries carried several AMR genes.As shown in Table 6, the hybrid plasmids of

Prevalence of colistin-resistant E. coli carrying mcr
This study revealed a high carriage rate of approximately 70% of colistin-resistant mcr-E.coli in the residents of all three communities, Santo Domingo and Puyo in Ecuador and Nguyen Xa in Vietnam.Recent reports by other groups showed the prevalence of mcr-E.coli to be 14%-38% in human stool samples from Vietnam [24,25] and Bolivia in South America [26].
Although it is lower than the carriage rate observed in the present study, it is presumed that this may be due to differences in the experimental conditions and participants.In particular, the selection medium used for colistin-resistant bacteria may greatly affect the carriage rate of resistant bacteria in the stools.Nevertheless, such a high colistin-resistant E. coli carriage found among the residents in the study was associated with a high prevalence of colistin-resistant E. coli in the livestock of the same area [14,16], which might be relevant with regards to the One Health [27].The similar levels of colistin-resistant E. coli in these geographically and culturally different communities/regions indicate that the wide dissemination of colistin-resistant E. coli carrying mcr in communities of low-and middle-income countries is a major threat to global public health.

Phylogenetic homology of colistin-resistant E. coli isolates
The phylogenetic homology of colistin-resistant E. coli disseminated among the communities and countries was assessed using the genome sequences of isolates.The Ecuador and Vietnam isolates were clustered into five major clades.However, the number of isolates in each clade and the distribution of sequence types were different between the two countries.
In Ecuador isolates, all the clades included mixed types of STs, but ST48 and ST10 were dominant among them.In addition, the mcr-1-carrying IncI2 plasmids were frequent among the isolates and distributed in various clades, as opposed to being concentrated in specific clades.The Ecuador isolates were from two geographically distinct communities within the same country-one in the Pacific coast and the other in the Amazon region-but each isolate did not fall into a specific clade of the community.Furthermore, the mcr-carrying plasmid IncI2 was widely distributed without bias in a specific clade.These results indicate that there are inter-regional social interactions between the communities.In contrast, in Vietnam, three of the five clades consisted of a single ST.In addition, the mcr-carrying plasmid IncI2 was rare in Vietnam isolates.Thus, although two communities in Ecuador and one community in Vietnam were assessed, the STs included in the clade of each country were different.In Vietnam, the spread by specific STs was dominant over the spread by mcr-carrying plasmids.It is unclear whether such differences between the two countries are caused by sociocultural differences or other factors.In this regard, it was reported that ST10, ST48, and ST165 were the common ST-types of colistin-resistant E. coli with mcr-1, and among them, ST10 was the most dominant group and was the main contributor to the dissemination of colistin-resistant E. coli [28,29].Besides relating to the colistin resistance, ST10 was also observed as the most common clone producing many extended-spectrum beta-lactamase (ESBL) types in E. coli, especially bla CTX-M [30][31][32][33].In the present study, ST10 was found in limited frequency in both Vietnam and Ecuador isolates, which confirmed that this strain has spread globally.Conversely, the ST48 clone has recently been identified to carry the bla KPC gene, which is associated with carbapenem resistance [34,35].The spreading of common ST clones carrying multi-AMR genes poses a significant challenge in controlling the antimicrobial resistance of bacteria.
When we analyzed the ST-type of all the mcr-E.coli isolates from Ecuador and Vietnam together, at least some phylogenies, including ST10, ST48, and ST206, were observed in both countries.This means that some lineages of E. coli carrying mcr are common host strains worldwide.This observation suggests that the mcr-plasmids may have spread to the endemic ST-type E. coli in these communities.That is, the wide dissemination of colistin-resistant E. coli in local communities is the result of the spread of mcr-carrying plasmids to endemic E. coli clones rather than the spread of mcr-carrying E. coli clones themselves.

Location and structure of mcr-transposon
The analysis of mcr location in the isolates from both Vietnam and Ecuador showed that the prevalence of chromosomal mcr in Ecuador was significantly lower than that in Vietnam.In particular, no chromosomal mcr was found in the Puyo isolates from Ecuador.Such differences in the chromosomal mcr frequency presumably indicate differences in the exposure of these organisms to colistin.The prevalence of chromosomal mcr in Vietnam isolates of this study is in line with the previous findings of another group that showed that 26% of Vietnam isolates carried mcr-1 on their chromosomes [24].This high prevalence of chromosomal mcr indicates the stable colistin resistance of E. coli.On the contrary, the data on mcr location in E. coli isolated from humans and animals in Ecuador is limited.In a study on characteristics of the mcr gene among E. coli from waters and sediments in Ecuador, only 1 of 459 Enterobacteriaceae bacteria carried chromosomal mcr [36].
The structure of the mcr-transposon Tn6330 has also contributed to the stability and transfer of mcr [9].That is, the mcr-transposon is considered to be stabilized by the structure in which insertion sequences (ISApl1) have been lost, although the transfer ability of mcr is limited [9,12].Interestingly, in the Vietnam isolates, all the mcr-transposons on the plasmids lost both or one of the ISs, whereas most of the Ecuador isolates had intact transposons that retained the IS.These results indicate that the mcr-transposons on the plasmids of Ecuador isolates may be progressing toward stabilization, whereas the mcr on the plasmids of Vietnam isolates are almost stabilized.The cause of these differences is unknown, but it probably depends on how long the community was exposed to colistin, such as the region-wide period of colistin use in domestic livestock.Regarding a report on antibiotic use and resistance in Vietnam, antibiotics were frequently used in animals with 70% of pharmaceutical products.Antibiotics were used not only in land-based livestock but also in aquaculture.In particular, colistin contributed to 5% of antibiotics used [37].Noteworthy, in the results of an updated study about colistin-based drug (CBD) used in chicken farms in Nguyen Xa, Vietnam, more than half of local farmers preferred using CBD than other antibiotics [38].Whereas the data on the consumption of antibiotics used in humans and animals was lacking in Ecuador [39].The ban on colistin for use in animal husbandry has been implemented in Ecuador since 2019, the effectiveness of this ban on colistin in Ecuador was discussed [40,41].In other words, colistin use in Vietnam may have been prevalent for a longer period than in Ecuador.This may also be the reason in the case of chromosomal mcr.
The chromosomal insertion site of the mcr-transposon is a crucial factor related to the mcr-1 insertion.An AT-rich region has a preference for the insertion site of the mcr-transposon, and the transposition forms a 2-bp duplication [42,43].The AT-rich region is the site where DNA replication and synthesis are initiated; it is also a low thermodynamic stability region [44].In this study, it was shown that the ISApl1 insertion sites of most of Ecuador and Vietnam isolates were surrounded by AT-rich regions.This finding is consistent with the results of previous studies [42,43].

mcr-carrying plasmid
The mobile colistin resistance genes, mcr, mediated by plasmids is the mechanism of concern leading to the wide dissemination of colistin resistance bacteria worldwide [45].The mcr-carrying plasmids are characterized by the Inc-type, and the main Inc-type is different depending on the site of isolation.This study showed that the Inc-type of the mcr-carrying-plasmids was diverse in both Ecuador and Vietnam isolates, but most of the plasmids belonged to four types -IncI2, IncP1, and IncX1/X4.In particular, the mcr-carrying IncI2-plasmid was remarkably prevalent in both Ecuador communities.In this regard, it was established that IncI2, IncX4, and IncHI2 contributed to more than 90% of the published mcr-1-carrying-plasmid types and were the dominant types [12,28].IncI-plasmids, including IncI2, are known to have low copy numbers; the host range is narrow [46,47], and they can transfer among different bacteria within the Enterobacteriaceae family, with E. coli as the predominant carrier [48].Therefore, the dominance of IncI2 in mcr-carrying plasmids in Ecuador is likely a dominant factor contributing to the transfer of mcr.In contrast, the X1/X4 Inc-types were preferentially identified among the Vietnam isolates.Five sub-types of the IncX-plasmids, IncX1-IncX5, have been identified [49].Within the IncX-group, the IncX4 subtype is the most predominant [50].Besides IncI2, IncX4 plasmids are also considered to be relevant in the spread of mcr-1 genes in the family Enterobacteriaceae [51,52].IncX1/X4 plasmids carrying mcr were prevalent in Vietnam isolates, unlike IncI2 in Ecuador isolates, indicating that the Inc-type plasmids involved in community mcr transmission were different and distinctive among communities.
The results of the comparative analysis of mcr-plasmids between isolates from both countries showed that the structures of mcr-carrying-IncI2-plasmids in both Ecuador and Vietnam isolates were almost the same in size and constituent genes.Similar results were also obtained with other Inc-type plasmids carrying mcr.Although it is known that the same Inc-types of mcr-plasmids carried by different strains are similar [53,54], this is the first report confirming this result among strains collected across countries.This suggests that mcr-carrying plasmids, such as the mcr-IncI2 plasmid, are spreading across communities and even countries while transferring among different phylogenetic host bacteria.

AMR genes on plasmids carrying mcr
The presence of other AMR genes on mcr-plasmids increases the risk of mcr-bacteria dissemination among communities.By detecting AMR genes on plasmids carrying mcr, we showed that more than half of the mcr-plasmids did not carry other resistance genes.These findings are consistent with a previous report that states that the co-existence of other AMR genes with mcr-1 on the plasmids occurs at a low percentage [55].Contrastingly, nearly half of the remaining plasmids, especially the hybrid plasmids, carried many AMR genes besides mcr.However, the AMR genes carried by the hybrid plasmid differed between Vietnam and Ecuador isolates, indicating regional characteristics.For example, aminoglycoside resistance genes were frequently present in the hybrid plasmids with mcr in the Vietnam isolates, whereas trimethoprim, streptomycin/spectinomycin, and chloramphenicol resistance genes were major AMR genes in the Ecuador isolates.The reasons for the differences in the AMR genes carried on mcr-plasmids of the isolates between the two countries are unknown, but it seems to be due to differences in the antibiotics frequently used in particular areas.Since the actual situation of antibiotic use in these areas is unknown, it remains a topic for future study.Nevertheless, mcrplasmids carrying AMR genes are a major public health risk.

Fig 1 .
Fig 1.The SNP phylogenetic tree of colistin-resistant E. coli isolates obtained from residents in (A) Ecuador and (B) Vietnam.The scale bar represents the nucleotide substitutions per site.The isolate IDs shown in blue and red in (A) indicate those isolated from Santo Domingo and Puyo, respectively.The Inc-type legend indicates isolates carrying the IncI2 plasmid with mcr-1.The mcr location legend indicates the presence of chromosomal mcr.https://doi.org/10.1371/journal.pone.0293940.g001

Table 3 . Inc-type of plasmid carrying mcr-1. Country Community No. of isolates tested No. of plasmids Plasmid Inc-type
a Part of the data is quoted from the work of Yamaguchi et al. [23].https://doi.org/10.1371/journal.pone.0293940.t003